【作者】 吕顺莉；

【导师】 金震东； 李兆申； 高军； 杜奕奇；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 胰腺癌(pancreatic carcinoma)是消化系统较常见、恶性程度较高的肿瘤,进展迅速,手术切除率低,预后极差,近几年来发病率在国内外均有逐年上升趋势。由于胰腺癌在临床上缺乏特异表现,在就诊时3／4的病人已属晚期,确诊病例手术可切除率仅为4%～27%,世界上每年有胰腺癌新发病例约20万人,占全部恶性肿瘤发病的2%,其死亡率与发病率十分接近,每年死亡人数约19.6万人,5年生存率<5%。因此,深入研究胰腺癌的分子发病机制显得十分必要。表观遗传学是指在不改变DNA序列的前提下调控基因的表达。表观基因组学则是在基因组水平上对表观遗传学改变的研究。表观遗传学包括DNA甲基化、组蛋白修饰以及由非编码RNA调控的mRNA表达等。近年的大量研究表明,遗传学中基因启动子区甲基化程度的改变对疾病的发生发展起重要作用。甲基化对基因的表达有着重要的调控作用,另外它还同基因组印记、女性X染色体的基因灭活、细胞增殖、分化发育、肿瘤的发生和发展以及遗传的不稳定性等密切相关。随着深入的研究,发现DNA甲基化成为阐明正常和病理基因表达现象的重要机制,且在肿瘤的早期诊断和病人愈后判断方面也有广阔的应用前景。基于上述研究现状,本课题首先利用甲基化基因芯片技术进行胰腺癌甲基化异常基因的筛查,发现新的胰腺癌异常甲基化基因PCDH8 (protocadherin8)和LRRC55 (leucine rich repeat containing 55),为胰腺癌的早期诊断和防治提供理论基础。进一步进行了两条基因在胰腺癌中的甲基化、表达等方面的研究,初步探讨了两条基因的功能。一、胰腺癌异常甲基化基因的芯片筛查目的：本研究利用以微阵列技术为基础的甲基化基因芯片,筛查胰腺癌异常甲基化基因,为胰腺癌的早期诊断找出更好的甲基化基因诊断靶标。方法：以4例胰腺癌及癌旁组织为研究对象,抽提并超声处理DNA,甲基化富集,将拟行对比分析的DNA样品分别行Cy3、Cy5标记,与甲基化基因芯片进行杂交,洗片,用激光扫描仪对芯片进行扫描,并用NimbleScan软件分析Cy3和Cy5的强度,用SignalMap软件以Cy3和Cy5比值大于2为标准筛选胰腺癌高甲基化基因,并在不同组织间比较找出胰腺癌高甲基化基因,同时进行胰腺癌高甲基化基因的定位分析。结果：初步研究了胰腺癌及癌旁组织样品DNA与甲基化芯片杂交结果,通过对比分析发现其中第11、13号染色体CpG岛有较多的CpG甲基化区域高甲基化,分析结果为第11号染色体LRRC55和第13号染色体PCDH8。结论：通过甲基化基因芯片筛查,发现高甲基化的CpG区域并定位,为寻找胰腺癌异常高甲基化基因奠定了基础。二、胰腺癌中PCDH8基因的甲基化、杂合性缺失和表达的检测目的：检测PCDH8基因在胰腺癌中甲基化状态、杂合性缺失状态、mRNA及蛋白表达。方法：首先收集2株胰腺癌细胞株、2例胰腺及癌旁组织、2例正常胰腺组织作为研究对象,健康成人外周血液标本2例作为对照组,应用BSP克隆测序法检测PCDH8基因启动子区CpG岛甲基化情况。通过MSP和QMSP法检测PCDH8基因在6株胰腺癌细胞株和64例人胰腺癌及相应癌旁胰腺组织中的甲基化状态。采用PCR+STR法检测PCDH8基因在36例人胰腺癌及相应癌旁胰腺组织中的杂合性缺失状态。应用实时定量PCR方法检测59例人胰腺癌及相应癌旁胰腺组织中PCDH8 mRNA的表达和6株胰腺癌细胞株在DNA甲基化转移酶抑制剂(5Aza-dC)处理前后mRNA的表达情况。利用免疫组织化学的方法检测42例人胰腺癌组织及癌旁胰腺组织和6例正常胰腺组织中PCDH8蛋白的表达部位及表达水平。结果：BSP克隆测序法检测结果表明PCDH8基因在2株胰腺癌细胞株和2例胰腺癌组织中呈现高甲基化状态(平均甲基化百分数分别为95%,52%),而在2例正常胰腺组织、2例癌旁组织和2例外周血液中为低甲基化状态(平均甲基化百分数分别为4%,6%,5%)。通过MSP法检测PCDH8基因在Pancl、BxPC3和CFPAC胰腺癌细胞株中部分甲基化,AsPC1、PaTu8988和SW1990胰腺癌细胞株完全甲基化；QMSP法检测胰腺癌组织甲基化率高于癌旁组织,甲基化率分别为0.015(0,0.074),0.0004(0,0.025)(P<0.05)。杂合性缺失检测结果D13s1228位点无杂合性缺失,D13s270位点杂合性缺失率为41%(13／32)。实时定量PCR检测结果为胰腺癌组织mRNA相对表达量(RQ)低于癌旁组织,RQ分别为0.03(0.01,0.14),0.09(0.02,0.35)(P<0.05)。应用DNA甲基化转移酶抑制剂处理人胰腺癌细胞株,处理后mRNA相对表达量明显高于处理前。PCDH8基因在各细胞株表达情况：AsPC-1在处理前无表达,5Aza-dC处理后RQ为12.55；BxPC3在处理前无表达,5Aza-dC处理后RQ为5.59；Panc1在处理前RQ为1.10,5Aza-dC处理后RQ为6.76；SW1990在处理前RQ为0.004,5Aza-dC处理后RQ为5.24；PaTu8988在处理前RQ为0.001,5Aza-dC处理后RQ为116.08；CFPAC在处理前RQ为0,5Aza-dC处理后RQ为592.63。免疫组织化学检测结果提示,PCDH8蛋白在胰腺癌组织中恶变的胰腺导管上皮细胞胞浆少量表达(9／42),间质细胞胞浆大部分表达(28／42)；在癌旁组织中胰腺导管胞浆少量表达(2／42),间质细胞胞浆少量表达(5／42)；在正常胰腺组织中胰腺导管腺泡无表达。胰腺癌组织中间质的表达高于癌旁组织,有统计学差异(P<0.05)。结论：PCDH8基因在胰腺癌中呈高甲基化状态；PCDH8基因在人胰腺癌组织中处于低表达状态；胰腺癌中PCDH8基因存在杂合性缺失；PCDH8基因启动子甲基化参与其基因的表达调控,基因启动子高甲基化是导致在胰腺癌中的低表达的重要原因：免疫组织化学检测提示胰腺导管癌细胞少量阳性,癌旁组织与癌组织的间质阳性存在差异。三、胰腺癌中LRRC55基因的甲基化和表达的检测目的：检测LRRC55基因在胰腺癌中甲基化状态、mRNA及蛋白表达。方法：首先收集2株胰腺癌细胞株、2例胰腺癌及癌旁组织、2例正常胰腺组织作为研究对象,健康成人外周血液标本2例作为对照组,应用BSP克隆测序法检测LRRC55基因启动子区CpG岛甲基化情况。通过QMSP检测LRRC55基因在64例人胰腺癌及相应癌旁胰腺组织中的甲基化状态。应用实时定量PCR方法检测42例人胰腺癌及相应癌旁胰腺组织中LRRC55 mRNA的表达。利用免疫组织化学的方法检测31例人胰腺癌组织及癌旁胰腺组织和6例正常胰腺组织中LRRC55蛋白的表达部位及表达水平。结果：BSP克隆测序法检测结果表明LRRC55基因在2株胰腺癌细胞株和2例胰腺癌组织中呈现高甲基化状态(平均甲基化百分数分别为71%,53%),而在2例正常胰腺组织、2例癌旁组织和2例外周血液中为低甲基化状态(平均甲基化百分数分别为11%,8%,9%)。通过QMSP法检测LRRC55基因胰腺癌组织甲基化率高于癌旁组织,甲基化率分别为0.016(0.0002,0.077),0.003(0,0.078),两组之间存在统计学差异(P<0.05)。实时定量PCR检测结果为胰腺癌组织mRNA相对表达量低于癌旁组织,RQ分别为0.14(0,1.03),0.99(0.19,3.69),两组之间表达量有统计学差异(P<0.05)。免疫组织化学检测结果提示,LRRC55蛋白在胰腺癌组织中恶变的胰腺导管上皮细胞胞浆大部分表达(27／30)；在癌旁组织中胰腺导管胞浆部分表达(20／30),腺泡细胞胞浆大部分表达(17／23)；在正常胰腺组织中胰腺导管和腺泡细胞无表达。胰腺癌组织中癌细胞的表达与癌旁组织无统计学差异(P>0.05)结论：LRRC55基因在胰腺癌中呈高甲基化状态；LRRC55基因在人胰腺癌组织中处于低表达状态；免疫组织化学检测提示胰腺导管癌细胞多数阳性,癌旁组织与癌组织之间无差异。通过上述研究,本课题得出以下结论：1、DNA甲基化芯片可为胰腺癌筛查出新的候选致病相关基因；2、候选基因PCDH8的启动子区高甲基化和杂合性缺失共同参与胰腺癌的发病；3、候选基因LRRC55的启动子区高甲基化参与胰腺癌的发病。更多还原

【Abstract】 Pancreatic carcinoma is a kind of more common and malignant tumor in digestive system. It’s attack rate is rising year by year in recent years. The more patients usually were in advanced stage when they were diagnosed, because of no specific clinical presentation, high malignancy, and easy to metastasis. The rate of operation resection of diagnosed patients is only 4%～27%. It was reported that there were approximately 200 thousand new cases of pancreatic cancer which accounted for 2%of total malignancies. The incidence of pancreatic cancers was near to mortality. The survival rate of five years is less than 5%. However, very little is known about the exact molecular mechanism in the formation of pancreatic carcinoma.Epigenetic refers to the study of mechanisms that control somatically heritable gene expression states without changes in the underlying DNA sequence. Epigenetic mechanisms include DNA methylation, histone modifications such as acetylation and methylation, and control of mRNA expression by non-coding RNAs. Recently bulk research indicate that the change of methylation degree in genic promoter region plays an important role in the process of disease occurrence and development. Methylation plays an important regulating role in genetic expression, in addition it has a close correlation with the imprinting of genetic group, genetic deactivation of female X chromosomal, cell proliferation, differentiation development, occurrence and development of tumors as well as genetic instability. DNA methylation plays a critical role in the development of mammals and the formation of neoplasm, and is potentially useful to the diagnosis and therapy of neoplasm.In this study, utilizing methylation gene microarray technique to screen different methylation gene among pancreatic cancer and normal pancreas, we found new specific methylated genes, PCDH8 (protocadherin 8) and LRRC55 (leucine rich repeat containing 55) for pancreatic cancer. This will create a better condition for pancreatic cancer’s early diagnosis and treatment. And we detected the molecular mechanism of PCDH8 and LRRC55 in pancreatic cancers and investigated the function of two genes.1. Microarray screening for aberrant methylation gene in pancreatic cancerObjective:This research utilized methylated genic microarray to screen specific hpermethylated aberrant gene of pancreatic cancer, in order to find better methylated genic diagnosis target for early diagnosis of pancreatic cancer.Methods:To take 4 pancreatic cancer and tissues surrounding cancer as research objects, extract all kinds of tissues DNA, prepared composite DNA samples of normal pancreatic tissue, treated DNA samples with ultrasound, enriched methylation, marked DNA samples with Cy3、Cy5, hybridizated with methylated genic microarray, wash slices, scan microarray with laser scanner, analyzed intensity of Cy3 and Cy5 with NimbleScan software, used Signal Map software according to Cy3:Cy5>2 as standard to screen high methylated gene of pancreatic cancer, compared in different tissues in order to find high methylated gene in pancreatic cancer, but no or hypomethylated gene in normal or pancreatitis tissues, meanwhile located hypermethylated abbrrent gene of pancreatic cancer.Results:We initially analyized the different aberrant hypermethylation gene between cancer tissues and composite sample DNA of normal pancreatic tissues after their hybridization with methylated gene microarray respectively. we found that there were high methylated aberrant genic regions in 11 and 13 chromosomes. They were PCDH8 and LRRC55.Consults:We found some hypermethylated aberrant CpG regions by methylated gene microarray screening. This will establish the foundation for looking for specific hypermethylated aberrant gene in pancreatic cancer.2. Expression patterns of PCDH8 gene in normal pancreas, and primary pancreatic adenocarcinomasObjective:To detect the expression of PCDH8 gene, involving the mRNA level, the protein level, loss of heterozygosity(LOH),and methylation status in pancreatic tissues.Methods:To detect the PCDH8 methylation status in 64 pancreatic cancer tissues and adjacent pancreatic tissues,6 pancreatic cancer cell lines by real-time quantitative MSP and BSP. We detect the PCDH8 mRNA in 59 pancreatic cancer tissues and adjacent pancreatic tissues by real-time PCR. We used PCR and STR methods to detect LOH in 36 pancreatic cancer tissues and adjacent pancreatic tissues. Six pancreatic cancer cell lines, were treated by DNA methyl-transferase inhibitor 5-aza-dC. We analyzed the changes of the relative quantity of PCDH8 mRNA level with and without treatment. We analyzed the localization and the level of PCDH8 protein in 42 pancreatic cancer tissues, adjacent pancreatic tissues and 8 normal pancreatic tissues by immunohistochemistry (IHC).Results:MSP analysis revealed the promoter region of PCDH8 gene to have an unmethylated status in the normal pancreatic tissues or a strong unmethylation with the weak methylation, while PCDH8 was frequently hypermethylated in pancreatic cancer tissues and cell lines. And LOH at D13s270 was 41%(13/32). Quantitative RT-PCR revealed PCDH8 was expressed in both normal and tumor tissue from the same patient, but the expression level in the pancreatic cancer tissues was significantly lower than that in the paired adjacent normal tissues(P<0.05). Treatment with 5-Aza-dC could increase the expression of PCDH8 mRNA relative quantities(RQ). In AsPC-1 cells, PCDH8 didn’t express without any treatment,12.55 after 5-Aza-dC treatment. In BxPC-3 cells, PCDH8 didn’t express without any treatment,5.59after 5-Aza-dC treatment. In Panc-1 cells, PCDH8 RQ were 1.10 without any treatment,6.76 after 5-Aza-dC treatment. In SW1990 cells, PCDH8 RQ were 0.004 without any treatment,5.24 after 5-Aza-dC treatment. In IHC, PCDH8 expressed in cancer cells(9/42), while almost all the pancreatic ducts showed negative in normal tissues.Consults:Comparing with the adjacent normal tissues, PCDH8 gene was down-regulated in pancreatic cancer tissues. The hypermethylation of PCDH8 gene promoter regulated the expression of its mRNA. The hypermethylation of PCDH8 gene promoter regulated the expression of its mRNA, and was an important reason that resulted in down-regulation in pancreatic cancer. IHC results suggested that it was negative staining in most pancreatic cancer cells.3. Expression patterns of LRRC55 gene in normal pancreas, and primary pancreatic adenocarcinomasObjective:To detect the expression of LRRC55 gene, involving the mRNA level, the protein level, and methylation status in pancreatic tissues.Methods:To detect the LRRC55 methylation status in 64 pancreatic cancer tissues and adjacent pancreatic tissues,6 pancreatic cancer cell lines by real-time quantitative MSP and BSP. We detect the LRRC55 mRNA in 42 pancreatic cancer tissues and adjacent pancreatic tissues by real-time PCR. We analyzed the localization and the level of LRRC55 protein in 30 pancreatic cancer tissues, adjacent pancreatic tissues and 8 normal pancreatic tissues by immunohistochemistry (IHC).Results:MSP analysis revealed the promoter region of LRRC55 gene to have an unmethylated status in the normal pancreatic tissues or a strong unmethylation with the weak methylation, while LRRC55 was frequently hypermethylated in pancreatic cancer tissues and cell lines. Quantitative RT-PCR revealed LRRC55 was expressed in both normal and tumor tissue from the same patient, but the expression level in the pancreatic cancer tissues was significantly lower than that in the paired adjacent normal tissues(P<0.05). In IHC, LRRC55 expressed in cancer cells(27/30), and the pancreatic ducts(20/30).Consults:Comparing with the adjacent normal tissues, LRRC55 gene was down-regulated in pancreatic cancer tissues. The hypermethylation of LRRC55 gene promoter regulated the expression of its mRNA. IHC results suggested that it expressed in most pancreatic cancer cells.In conclusion:1. Microarray screening for hypermethylation abbrrent gene in pancreatic cancer is an effective method.2. The hypermethylation of the promoter and LOH both affected human pancreatic cancer.3. The hypermethylation of the promoter of LRRC55 gene was one of the reasons that resulted in the down-regulation of LRRC55 gene in pancreatic tissues更多还原